Project Summary/Abstract Kidney cancer, or renal cell carcinoma (RCC), is among the ten most prevalent malignancies in the United States, and has exhibited an increasing incidence rate in both men and women since 2001. The most common subtype of RCC is ?clear cell? RCC (ccRCC, 75% of all cases). ccRCC is characterized by chemotherapy and radiation resistance; while surgical resection of early stage disease can be curative, five year relapse rates approach 40%, with the majority of these cases developing metastases. Of note, ccRCCs lack common genetic abnormalities observed in many other human cancers, including mutations in the PTEN, AKT, TP53, and KRAS loci, hindering successful treatment of ccRCC by corresponding targeted therapies. In contrast, ccRCCs feature consistent metabolic abnormalities, such as highly elevated glycogen and fat deposition. These metabolic disorders are associated with normoxic stabilization of hypoxia-inducible factors (HIFs), secondary to von Hippel-Lindau (VHL mutations) that occur in > 90% of ccRCC tumors. Because Vhl ablation in mouse kidney fails to induce ccRCC formation, additional oncogenic changes may be required. By integrating exome sequencing, copy number variation, transcriptomic, and metabolomic data, we identified multiple metabolic enzymes as universally depleted in all ccRCC tumors (an otherwise genetically heterogeneous disease). The first pathway involves decreased gluconeogenesis and glycogen storage, as regulated by fructose-1,6-bisphosphatase (FBP1). FBP1 loss significantly correlates with advanced tumor stages and poor patient survival, consistent with its tumor suppressor functions in inhibiting glycolysis, NADPH production, and nuclear HIF activity. The second most down-regulated metabolic pathway in ccRCC is the urea cycle, including the argininosuccinate synthase 1 (ASS1), argininosuccinate lyase (ASL), and arginase 2 (ARG2) enzymes. Of note, FBP1, ASS1, and ASL have both catalytic activity-dependent and catalytic activity-indendent, or structural roles. For example, FBP1 exhibits both cytoplasmic metabolic activity and nuclear transcriptional effects on HIF and other nuclear proteins. We propose to investigate the unprecedented, non-catalytic roles of these enzymes in ccRCC and other cancers. ccRCC also exhibit unusually high numbers of lipid droplets, organelles which store triglycerides and cholesterol esters, whose overproduction is a hallmark of this disease. Delineating the molecular mechanisms by which changes in gluconeogenesis, the urea cycle, and lipid homeostasis alter ccRCC tumor metabolism will provide new therapeutic avenues to target a majority of patients diagnosed with this kidney cancer subtype. The results obtained from ccRCC will also be applied to other malignancies, including soft-tissue sarcoma, hepatocellular carcinoma, and Burkitt's lymphoma, which appear to engage in highly similar metabolic reprogramming.